Fig. 1. Clusterin potentiates seeding of Tau aggregates.
a Workflow of the seeding experiment. At the times indicated, samples were removed from TauRD aggregation reactions with or without Clu, and added with or without transfection reagent (lipofectamine) to reporter cells co-expressing TauRD fused to the FRET pair of YFP and mTurquoise2 (TauRD-YT). The fraction of cells containing FRET-positive (pos.) aggregates was quantified by flow cytometry. b TauRD amyloid formation in aggregation reactions of 10 µM TauRD without (black) or with equimolar Clu (red) as monitored by ThT fluorescence. Averages ± SEM (n = 10 independent experiments). arb.units, arbitrary units. c Effect of Clu on the formation of seeds that induce aggregation of endogenous TauRD in cells. Seed formation was analyzed as described in (a). Reporter cells were transfected with aggregation reactions containing 14 ng (solid lines) or 0.05 ng (dashed lines) TauRD and 51 ng (solid lines) or 0.2 ng (dashed lines) Clu, respectively (molar ratio Clu:TauRD 1:1). Averages ± SEM (n = 3 independent experiments). d Representative fluorescence microscopy images of TauRD-YT cells seeded with TauRD (14 ng TauRD) from the plateau phase of aggregation (TauRD, 1 h reaction time; TauRD/Clu, 6 h reaction time (b)). TauRD-YFP and DAPI nuclear staining signals are shown in yellow and blue, respectively. Scale bars, 20 μm for overview panels and 10 μm for insets. e Fold change of seeding potency of TauRD aggregation reactions containing Clu (red) compared to control reactions without Clu (gray). Bar graphs represent the average slope ± SEM (n = 3 independent experiments) from the linear regression analyses described in Supplementary Fig. 2e. ***p < 0.001 (p = 1.2 × 10−4) by two-tailed Student’s t-test. f Effect of Clu on seeding potency of TauRD-YT cell lysates containing TauRD-YT aggregates. Whole-cell lysates of FRET-positive (pos.) TauRD-YT cells were incubated with or without Clu. Fold change of TauRD seeding potency expressed per ng of TauRD-YT in cell lysates upon treatment with increasing Clu (molar ratios TauRD-YT:Clu 1:2, 1:10 and 1:20). Bar graphs represent the average slope ± SEM (n = 3 independent experiments) from the linear regression analyses shown in Supplementary Fig. 4e. ***p < 0.001 (-Clu vs. TauRD-YT:Clu 1:10 p = 8 × 10−4; -Clu vs. TauRD-YT:Clu 1:20 p = 1.1 × 10−8) by one-way ANOVA with Bonferroni post hoc test.